Recent years have brought amazing progress in the areas of molecular biology and genetics. The findings accumulated in such fields have not only contribution to the chemical and physical elucidation of biological phenomena, but also great influence on the human being, especially on medical science and practice. As a consequence, the DNA medicine, which started from DNA investigation, is marking great steps toward clinical fields far beyond the expectation. As it is recently revealed that almost all diseases are related to DNA, diagnosis at gene level is gradually regarded to be indispensable.
It is believed that those diseases now generally referred to as gene diseases (molecular diseases) include almost all of the enzyme deficiencies which are long known as diseases caused by inborn errors of metabolism. Detection of gene mutation is quite effective for the diagnosis of such gene diseases.
When a large number of genotypes are present as in the case of human leukocyte antigen (HLA), which hold the key to success of bone marrow transplantation or organ transplantation, it is desired to conveniently and rapidly determine the gene type at a high specificity.
An exemplary method for detecting the gene mutation or determining the gene type is the method of Nicolas, J. C. et al. (EP-A 362042; Anal. Biochem. 205, 193 (1992)), which is a modification of ED-PCR detection system (Japanese Patent Application Kokai (JP-A) Nos. 314965/1989 and 252300/1989; J. Clin. Microbiol. 30, 1728 (1992)).
The method of Nicolas et al. is carried out as follows. First, a labeled standard DNA is prepared from a double stranded nucleic acid fragment including a mutation region to be detected by introducing biotin label in one strand and FITC label in the other strand of the nucleic acid fragment. The labeled standard DNA is then mixed with an excess amount of a sample DNA including an unlabeled nucleic acid fragment of the same region as the standard DNA, and the mixture is heated for denaturation and slowly cooled (competitive hybridization). When a fragment including a nucleotide sequence identical with that of the labeled standard DNA is present in the sample, strand exchange would occur between the double stranded labeled standard DNA and the double stranded sample DNA, resulting in the amount of the labeled standard DNA possessing both the biotin label and the FITC label being reduced from the initial amount. On the other hand, when a fragment including a nucleotide sequence which is partly different from that of the labeled standard DNA is present in the sample, strand exchange is unlikely to occur between the double stranded labeled standard DNA and the double stranded sample DNA and the initial amount of the labeled standard DNA would remain substantially unchanged. Briefly stated, this method determines whether or not a fragment including a nucleotide sequence identical with that of the labeled standard DNA is present, by observing a change of the initial amount of the labeled standard DNA added through the steps of mixing, denaturation and annealing (competitive hybridization).
Also, the inventors of the present invention proposed a nucleic acid-differentiation method which is an improvement over the method of Nicolas et al. (PCT/JP94/01106, Nucl. Acids. Rec. 22, 1541 (1994)). In this method, a primer including a detectable label and a site capable of binding with a solid phase carrier is used for the PCR amplification of a target nucleic acid to produce a labeled DNA (sample DNA); and contrary to the above-described method of Nicolas et al., at least an equimolar amount of unlabeled DNA specimen of the same region (standard DNA) is added to the thus obtained labeled DNA (sample DNA) for competitive hybridization. The degree of complementary strand exchange between said sample DNA and said standard DNA is measured utilizing said detectable label and said site capable of binding with the solid phase carrier, to thereby determine the identity of nucleic acids. This method enables detection of a minute amount of mutant gene and detection of the mutation present on only one of allele, which are difficult with the above-referred method of Nicolas et al., and further can detect the proportion of such mutation.
The nucleic acid-differentiation methods of detecting gene mutation or the like by employing the detection system of ED-PCR and combining it with competitive hybridization, inclusive of the method of Nicolas et al. and the method proposed by the present inventors, are generally referred to as PCR-PHFA, hereinafter.
When gene mutation is detected by the PCR-PHFA, the primer-binding site in the nucleotide sequence may be adequately selected, and a number of primers may be prepared from which the most suitable primer is selected.
The choice of the primer-binding site, however, would be limited when the information on the nucleotide sequence is limited, or when the sequence to be amplified is common to several genes. For example, in the case of determining the HLA type or the like, DNAs of various types should be amplified with one pair of primers for the sake of examination efficiency, and consequently, the choice of the primer binding sequence is limited to the sequence commonly found among the different DNA types. As a consequence, the site of nucleotide difference between particular types of DNAs can be situated quite near the primer binding site. Continuing further investigations, the present inventors found that, in such case, the nucleotide (sequence) to be differentiated is located near the terminal of the PCR amplification product and sometimes difficult to differentiate with the above-described PCR-PHFA.